Browsing by Author "Orio, P"

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    Apical sorting of a voltage- and Ca2+-activated K+ channel α-subunit in Madin-Darby canine kidney cells is independent of N-glycosylation
    (2000) Bravo-Zehnder, M; Orio, P; Norambuena, A; Wallner, M; Meera, P; Toro, L; Latorre, R; González, A
    The voltage- and Ca2+-acttiated K+ (K-V,K-Ca) channel is expressed in a variety of polarized epithelial cells seemingly displaying a tissue-dependent apical-to-basolateral regionalization, as revealed by electrophysiology. Using domain-specific biotinylation and immunofluorescence we show that the human channel K-V,K-Ca alpha -subunit (human Slowpoke channel, hSlo) is predominantly found in the apical plasma membrane domain of permanently transfected Madin-Darby canine kidney cells. Both the wild-type and a mutant hSlo protein lacking its only potential N-glycosylation site were efficiently transported to the cell surface and concentrated in the apical domain even when they were overexpressed to levels 200- to 300-fold higher than the density of intrinsic Slo channels. Furthermore, tunicamycin treatment did not prevent apical segregation of hSlo, indicating that endogenous glycosylated proteins (e.g., K-V,K-Ca beta -subunits) were not required. hSlo seems to display properties for lipid-raft targeting, as judged by its buoyant distribution in sucrose gradients after extraction with either detergent or sodium carbonate. The evidence indicates that the hSlo protein possesses intrinsic information for transport to the apical cell surface through a mechanism that may involve association with lipid rafts and that is independent of glycosylation of the channel itself or an associated protein. Thus, this particular polytopic model protein shows that glycosylation-independent apical pathways exist for endogenous membrane proteins in Madin-Darby canine kidney cells.
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    Neuropeptide Y induced inhibition of noradrenaline release in rat hypothalamus
    (1999) Bitran, M; Tapia, W; Eugenín, E; Orio, P; Boric, MP
    We aimed at characterizing the receptor subtype and the signaling pathway involved in the inhibitory effect of neuropeptide Y on the release of endogenous noradrenaline from rat hypothalamus. Slices of hypothalamus were stimulated with two trains of electrical pulses, and the release of noradrenaline and nitric oxide was measured, The electrical stimulation of hypothalamic slices induced a consistent release of both endogenous noradrenaline and NO. Neuropeptide Y inhibited concentration dependently the stimulated noradrenaline release. Similarly, agonists for neuropeptide Y Y-2, Y-2 and Y-5 receptors inhibited noradrenaline release, albeit with a potency lower than neuropeptide Y. GW1229, a selective neuropeptide Y Y-1 receptor antagonist counteracted the effect of neuropeptide Y, but not that of PYY-(3-36), an agonist active at neuropeptide Y Y-5 and Y-2 receptors. These results indicate that the inhibitory effect of neuropeptide Y is likely mediated by several receptor subtypes, including neuropeptide Y Y-2, Y-5 and possibly Y-2 receptors. One mu M NPY significantly enhanced NO release induced by the electrical stimulation. N-G-monomethyl-L-arginine, an inhibitor of nitric oxide synthase, abolished NO release and blocked the inhibitory effect of neuropeptide Y on noradrenaline release. We conclude that nitric oxide participates in the signaling pathway of neuropeptide Y in the rat hypothalamus. (C) 1999 Elsevier Science B.V. All rights reserved.